Allosteric Regulation of Glycogen Phosphorylase by Order/Disorder Transition of the 250' and 280s Loops.

Allostery is a fundamental mechanism of protein activation, yet the precise dynamic changes that underlie functional regulation of allosteric enzymes, such as glycogen phosphorylase (GlyP), remain poorly understood. Despite being the first allosteric enzyme described, its structural regulation is still a challenging problem: the key regulatory loops of the GlyP active site (250' and 280s) are weakly stable and often missing density or have large b-factors in structural models. This led to the longstanding hypothesis that GlyP regulation is achieved through gating of the active site by (dis)order transitions, as first proposed by Barford and Johnson. However, testing this requires a quantitative measurement of weakly stable local structure which, to date, has been technically challenging in such a large protein. Hydrogen-deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for studying protein dynamics, and millisecond HDX-MS has the ability to measure site-localized stability differences in weakly stable structures, making it particularly valuable for investigating allosteric regulation in GlyP. Here, we used millisecond HDX-MS to measure the local structural perturbations of glycogen phosphorylase b (GlyPb), the phosphorylated active form (GlyPa), and the inhibited glucose-6 phosphate complex (GlyPb:G6P) at near-amino acid resolution. Our results support the Barford and Johnson hypothesis for GlyP regulation by providing insight into the dynamic changes of the key regulatory loops.

"Grafting-To" Covalent Binding of Plasmonic Nanoparticles onto Silica WGM Microresonators: Mechanically Robust Single-Molecule Sensors and Determination of Activation Energies from Single-Particle Events.

We hereby present a novel "grafting-to"-like approach for the covalent attachment of plasmonic nanoparticles (PNPs) onto whispering gallery mode (WGM) silica microresonators. Mechanically stable optoplasmonic microresonators were employed for sensing single-particle and single-molecule interactions in real time, allowing for the differentiation between binding and non-binding events. An approximated value of the activation energy for the silanization reaction occurring during the "grafting-to" approach was obtained using the Arrhenius equation; the results agree with available values from both bulk experiments and ab initio calculations. The "grafting-to" method combined with the functionalization of the plasmonic nanoparticle with appropriate receptors, such as single-stranded DNA, provides a robust platform for probing specific single-molecule interactions under biologically relevant conditions.

Anomalous DNA hybridisation kinetics on gold nanorods revealed via a dual single-molecule imaging and optoplasmonic sensing platform.

Observing the hybridisation kinetics of DNA probes immobilised on plasmonic nanoparticles is key in plasmon-enhanced fluorescence detection of weak emitting species, and refractive index based single-molecule detection on optoplasmonic sensors. The role of the local field in providing plasmonic signal enhancements for single-molecule detection has been studied in great detail. Nevertheless, few studies have compared the experimental results in both techniques for single-molecule studies. Here we developed the first optical setup that integrates optoplasmonic and DNA-PAINT based detection of oligonucleotides to compare these sub-platforms and provide complementary insights into single molecule processes. We record the fluorescence and optoplasmonic sensor signals for individual, transient hybridisation events. The hybridisation events are observed in the same sample cell and over a prolonged time (i.e. towards high binding site occupancies). A decrease in the association rate over the measurement duration is reported. Our dual optoplasmonic sensing and imaging platform offers insight into the observed phenomenon, revealing that irreversible hybridisation events accumulate over detected step signals in optoplasmonic sensing. Our results point to novel physicochemical mechanisms that result in the stabilisation of DNA hybridisation on optically-excited plasmonic nanoparticles.

Sensing Enzyme Activation Heat Capacity at the Single-Molecule Level Using Gold-Nanorod-Based Optical Whispering Gallery Modes.

Here, we report a label-free gold nanoparticle-based single-molecule optical platform to study the immobilization, activity, and thermodynamics of single enzymes. The sensor uses plasmonic gold nanoparticles coupled to optical whispering gallery modes (WGMs) to probe enzyme conformational dynamics during turnover at a microsecond time resolution. Using a glucosidase enzyme as the model system, we explore the temperature dependence of the enzyme turnover at the single-molecule (SM) level. A recent physical model for understanding enzyme temperature dependencies (macromolecular rate theory; MMRT) has emerged as a powerful tool to study the relationship between enzyme turnover and thermodynamics. Using WGMs, SM enzyme measurements enable us to accurately track turnover as a function of conformational changes and therefore to quantitatively probe the key feature of the MMRT model, the activation heat capacity, at the ultimate level of SM. Our data shows that WGMs are extraordinarily sensitive to protein conformational change and can discern both multiple steps with turnover as well as microscopic conformational substates within those steps. The temperature dependence studies show that the MMRT model can be applied to a range of steps within turnover at the SM scale that is associated with conformational change. Our study validates the notion that MMRT captures differences in dynamics between states. The WGM sensors provide a platform for the quantitative analysis of SM activation heat capacity, applying MMRT to the label-free sensing of microsecond substates of active enzymes.

Optoplasmonic characterisation of reversible disulfide interactions at single thiol sites in the attomolar regime.

Probing individual chemical reactions is key to mapping reaction pathways. Trace analysis of sub-kDa reactants and products is obfuscated by labels, however, as reaction kinetics are inevitably perturbed. The thiol-disulfide exchange reaction is of specific interest as it has many applications in nanotechnology and in nature. Redox cycling of single thiols and disulfides has been unresolvable due to a number of technological limitations, such as an inability to discriminate the leaving group. Here, we demonstrate detection of single-molecule thiol-disulfide exchange using a label-free optoplasmonic sensor. We quantify repeated reactions between sub-kDa thiolated species in real time and at concentrations down to 100's of attomolar. A unique sensing modality is featured in our measurements, enabling the observation of single disulfide reaction kinetics and pathways on a plasmonic nanoparticle surface. Our technique paves the way towards characterising molecules in terms of their charge, oxidation state, and chirality via optoplasmonics.

Publisher Correction: Optoplasmonic characterisation of reversible disulfide interactions at single thiol sites in the attomolar regime.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Patellar button dissociation in a mobile-bearing LCS patellofemoral joint arthroplasty.

Patellofemoral arthroplasty is an effective treatment for isolated patellofemoral arthritis. Midterm results reveal a success rate of approximately 80% to 90% with modern designs. The reported failure mechanisms associated with patellofemoral arthroplasty include progressive tibiofemoral arthritis, patellar pain, catching or subluxation caused by soft-tissue imbalance, component malposition, and problematic designs. We present a previously unreported new complication of patellar button dissociation in a mobile-bearing LCS Patellofemoral Joint Replacement Prosthesis.

Cutaneous sensory loss following primary total knee arthroplasty. A two years follow-up study.

We examined 32 patients for skin flap numbness around their scars after primary total knee arthroplasty (TKA). All 32 patients were reviewed six weeks following surgery and two years thereafter. The aims of our study were to determine the natural history of cutaneous sensory loss, to identify the relationship with scar length and scar location, as well as the relationship between the size of the numb area and the severity of numbness and its recovery pattern. We also studied the relationship between numbness and other factors such as tourniquet time, lateral release and patella resurfacing. Twenty-six patients (81%) had lateral skin flap numbness; the other six patients (19%) had normal skin sensation around the scar. The size of the numb area was large in 19 patients (73%) out of 26. Our findings suggest more severe sensory loss is associated with larger numb area. Patients with a smaller numb area had less severe sensory loss. More laterally placed incisions were having better sensation (p = 0.00435). Patella resurfacing (p = 0.5) and lateral retinacular release (p = 0.10) were not significantly associated with the skin numbness. Fifty percent of our patients fully recovered from skin numbness at the end of two years. Patients with a small numb area had a more favourable outcome compared to those with a larger numb area. We conclude that permanent cutaneous sensory loss is not a universal occurrence following primary TKA; however a significant number of patients can be affected by this complication. All the patients should be warned about cutaneous numbness before the surgery.

Label-Free Optical Single-Molecule Micro- and Nanosensors.

Label-free optical sensor systems have emerged that exhibit extraordinary sensitivity for detecting physical, chemical, and biological entities at the micro/nanoscale. Particularly exciting is the detection and analysis of molecules, on miniature optical devices that have many possible applications in health, environment, and security. These micro- and nanosensors have now reached a sensitivity level that allows for the detection and analysis of even single molecules. Their small size enables an exceedingly high sensitivity, and the application of quantum optical measurement techniques can allow the classical limits of detection to be approached or surpassed. The new class of label-free micro- and nanosensors allows dynamic processes at the single-molecule level to be observed directly with light. By virtue of their small interaction length, these micro- and nanosensors probe light-matter interactions over a dynamic range often inaccessible by other optical techniques. For researchers entering this rapidly advancing field of single-molecule micro- and nanosensors, there is an urgent need for a timely review that covers the most recent developments and that identifies the most exciting opportunities. The focus here is to provide a summary of the recent techniques that have either demonstrated label-free single-molecule detection or claim single-molecule sensitivity.

Photonic sensing of arterial distension.

Most cardiovascular diseases, such as arteriosclerosis and hypertension, are directly linked to pathological changes in hemodynamics, i.e. the complex coupling of blood pressure, blood flow and arterial distension. To improve the current understanding of cardiovascular diseases and pave the way for novel cardiovascular diagnostics, innovative tools are required that measure pressure, flow, and distension waveforms with yet unattained spatiotemporal resolution. In this context, miniaturized implantable solutions for continuously measuring these parameters over the long-term are of particular interest. We present here an implantable photonic sensor system capable of sensing arterial wall movements of a few hundred microns in vivo with sub-micron resolution, a precision in the micrometer range and a temporal resolution of 10 kHz. The photonic measurement principle is based on transmission photoplethysmography with stretchable optoelectronic sensors applied directly to large systemic arteries. The presented photonic sensor system expands the toolbox of cardiovascular measurement techniques and makes these key vital parameters continuously accessible over the long-term. In the near term, this new approach offers a tool for clinical research, and as a perspective, a continuous long-term monitoring system that enables novel diagnostic methods in arteriosclerosis and hypertension research that follow the trend in quantifying cardiovascular diseases by measuring arterial stiffness and more generally analyzing pulse contours.

Implantable pulse oximetry on subcutaneous tissue.

Blood oxygen saturation is one of the most prominent measurement parameters in daily clinical routine. However up to now, it is not possible to continuously monitor this parameter reliably in mobile patients. High-risk patients suffering from cardiovascular diseases could benefit from long-term monitoring of blood oxygen saturation. In this paper, we present a minimally invasive, implantable patient monitor which is capable of monitoring vital signs. The capability of this multimodal sensor to subcutaneously determine blood pressure, pulse and ECG has been demonstrated earlier. This paper focuses on monitoring of blood oxygen saturation. Even though the signal amplitudes are much weaker than for standard extracorporeal measurements, photoplethysmographic signals were recorded with high quality in vivo directly on subcutaneous muscle tissue. For the first time, it has been shown that blood oxygen saturation can be measured with an implantable, but extravascular sensor. The sensor was implanted for two weeks in a sheep and did not cause any complications. This opens new perspectives for home monitoring of patients with cardiovascular diseases.

Radiative transport in large arteries.

A refined model for the photon energy distribution in a living artery is established by solving the radiative transfer equation in a cylindrical geometry, using the Monte Carlo method. Combining this model with the most recent experimental values for the optical properties of flowing blood and the biomechanics of a blood-filled artery subject to a pulsatile pressure, we find that the optical intensity transmitted through large arteries decreases linearly with increasing arterial distension. This finding provides a solid theoretical foundation for measuring photoplethysmograms.

Early results of whole femoral head allograft with articular cartilage for acetabular impaction grafting in revision hip replacements.

INTRODUCTION: Extensive bone loss associated with revision hip surgery is a significant orthopaedic challenge. Acetabular reconstruction with the use of impaction bone grafting and a cemented polyethylene cup is a reliable and durable technique in revision situations with cavitatory acetabular bone defects. Some use cancellous graft alone whilst others morselise the whole femoral head after removal of articular cartilage. This paper asks, if it really necessary to use pure cancellous graft? PATIENTS AND METHODS: Forty-two acetabular revisions using impacted morselized bone graft without removal of articular cartilage and a cemented cup were studied retrospectively. The mean follow-up was 3 years (range, 2-5.6 years). Clinical and radiographic assessment was made using the Oxford hip score, Hodgkinson's criteria (1988) for socket loosening and the Gie classification (1993) for evaluation of allograft consolidation and remodelling. RESULTS: Forty (95%) sockets were considered radiologically stable (type 0, 1, 2 demarcations) and two (5%) sockets were radiologically loose (Type 3 demarcation). There was no socket migration in our series. Twenty-seven(64%) cases showed good trabecular remodelling (grade 3). Twelve (29%) cases showed trabecular incorporation (grade 2). Only three (7%) cases showed poor allograft incorporation (grade 1). Average pre-operative Oxford hip score was 41 and postoperative hip score was 27. There have been no socket re-revisions (100% survival) at an average of 3 years. CONCLUSIONS: Early radiological and clinical survival results with retaining articular cartilage of femoral head allografts are similar and comparable to other major studies for acetabular impaction bone grafting in revisions. Minimal loss of allograft mass is 40% in obtaining pure cancellous graft. When there is a limited supply and demand of allograft, saving up to 40% of the material is a valuable and cost-effective use of scarce resources.